The invention relates to novel Exo1 and Exo2 proteins, nucleic acids and antibodies. The invention further relates to the use of bioactive agents such as Exo1 and Exo2 proteins, nucleic acids and antibodies for the diagnosis and treatment of disease.
Exocytosis is the fusion of secretory vesicles with the cellular plasma membrane, and has two main functions. One is the discharge of the vesicle contents into the extracellular space, and the second is the incorporation of new proteins and lipids into the plasma membrane itself.
Exocytosis can be divided into two classes: constitutive and regulated. All eukaryotic cells exhibit constitutive exocytosis, which is marked by the immediate fusion of the secretory vesicles after formation. Regulated exocytosis is restricted to certain cells, including exocrine, endocrine and neuronal cells. Regulated exocytosis results in the accumulation of the secretory vesicles that fuse with the plasma membrane only upon receipt of an appropriate signal, usually (but not always) an increase in the cytosolic free Ca2+ concentration. Regulated exocytosis is crucial to many specialized cells, including neurons (neurotransmitter release), adrenal chromaffin cells (adrenaline secretion), pancreatic acinar cells (digestive enzyme secretion), pancreatic xcex2-cells (insulin secretion), mast cells (histamine secretion), mammary cells (milk protein secretion), sperm (enzyme secretion), egg cells (creation of fertilization envelope) and adipocytes (insertion of glucose transporters into the plasma membrane). In addition, current theory suggests that the mechanisms of vesicle docking and fusion is conserved from yeast to mammalian brain.
Some insights into the process of regulated secretion at the molecular level have allowed the definition of G proteins as important regulators. Early experiments showed that non-hydrolyzable analogues of GTP could induce secretion in peritoneal mast cells (Fernandez et al., Nature 312:453 (1984)). More recently, a large body of evidence has been accumulating implicating small G proteins of the rab family as regulators in the fusion of secretory granules with plasma membranes during exocytosis. The rab proteins are a branch of the Ras superfamily of small GTPases. To date, the yeast rab family has 11 proteins, while over 40 ras-related proteins have been found in mammalian cells. The rab GTPases represent a diverse family of homologous proteins that are generally associated with the membrane of organelles in a wide variety of cells, where they regulate defined steps of intracellular membrane traffic (Zerial et al., Curr. Opin. Cell. Biol. 5:613 (1993)). An example of this are the rab3 subfamily proteins which have been found to have limited expression in regulated secretion-competent cells, and to be associated with synaptic or secretory granules, suggesting that they are involved in stimulus-secretion coupling (Lledo et al., Trends. Neurobiol. Sci. 17:426 (1994)). Further, overexpression of rab3D or its GTP binding mutant form (N1351) in the rat basophil line RBL leads to significant inhibition of IgE mediated exocytosis (Roa et al., J. Immunol. 159:2815 (1997)). Thus it appears that tissue/cell specific isoforms of rab proteins may play particular roles in regulated secretory responses.
In addition, disorders involving exocytosis are known. For example, inflammatory mediator release from mast cells leads to a variety of disorders, including asthma. Similarly, Chediak-Higashi Syndrome (CHS) is a rare autosomal recessive disease in which neutrophils, monocytes and lymphocytes contain giant cytoplasmic granules. Similar disorders have been described in mice, mink, cattle, cats, and killer whales, with the murine homolog of CHS (designated beige or bg) being the best characterized. See Perou et al., J. Biol. Chem. 272(47):29790 (1997) and Barbosa et al., Nature 382:262 (1996), both of which are hereby incorporated by reference.
Accordingly, the proteins involved in exocytosis are of paramount interest, and it is an object of the invention to provide Exo1 and Exo2 proteins and related molecules. It is a further object of the invention to provide recombinant nucleic acids encoding Exo1 and Exo2 proteins, and expression vectors and host cells containing the nucleic acid encoding the Exo1 and Exo2 proteins. A further object of the invention is to provide methods for screening for antagonists and agonists of Exo1 and Exo2.